Flow cytometry sorting of cells infected with African swine fever virus

Abstract

The African swine fever panzootic is continuing to spread, and the number of affected countries and material losses are increasing. In particular, India, Papua New Guinea, Malaysia, Greece and Bhutan joined the list of ASF infected countries in 2020–2021. The disease control is hindered by the lack of commercially available and effective vaccines, which, in its turn, is attributable to the insufficient knowledge of ASF pathogenesis and immune defense against the disease. The use of attenuated virus variants enables a thorough investigation of the factors influencing the virulence of African swine fever virus and the immune response to it. This involves the use of naturally attenuated virus variants, as well as of the variants attenuated by a long-term passaging of the virus in cell cultures. However, virulence heterogeneity characteristic of the ASF virus population, necessitates the additional selection of infected cells for the virus cloning. Conventional culture-based techniques for virus particle cloning are rather time- and labour-consuming; it is therefore appropriate to use flow cytometry cell sorting for the selection and cloning of virus infected cells with a view of selecting homologous virus lineages. The paper presents the results of sorting of African green monkey kidney cells (CV-1) and porcine bone marrow cells infected with African swine fever virus; the cells were sorted into the 96-well culture plates using a MoFlo Astrios EQ cell sorter in order to isolate a population of the virus originating from one infected cell. After the single cell sorting of the infected cell cultures into the 96-well plates, ASF positive cell detection rates in the plate wells were 30% for porcine bone marrow cells and 20% for CV-1.

Keywords

• african swine fever
• cytometry
• cell sorting
• cell culture